Spinal Cord (2009) 47, 2–35 & 2009 International Spinal Cord Society All rights reserved 1362-4393/09 $32.00 www.nature.com/sc

REVIEW

Autonomic assessment of animals with : tools, techniques and translation

JA Inskip1,2,4, LM Ramer1,2,4, MS Ramer1,2 and AV Krassioukov1,3

1International Collaboration on Repair Discoveries, University of British Columbia, Vancouver, British Columbia, Canada; 2Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada and 3Department of Medicine, Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, British Columbia, Canada

Study design: Literature review. Objectives: To present a comprehensive overview of autonomic assessment in experimental spinal cord injury (SCI). Methods: A systematic literature review was conducted using PubMed to extract studies that incorporated functional motor, sensory or autonomic assessment after experimental SCI. Results: While the total number of studies assessing functional outcomes of experimental SCI increased dramatically over the past 27 years, studies with motor outcomes dramatically outnumber those with autonomic outcomes. Within the areas of autonomic dysfunction (cardiovascular, respiratory, gastrointestinal, lower urinary tract, sexual function and thermoregulation), not all aspects have been characterized to the same extent. Studies focusing on bladder and cardiovascular function greatly outnumber those on sexual function, gastrointestinal function and thermoregulation. This review addresses the disparity between well-established motor-sensory testing presently used in experimental animals and the lack of standardized autonomic testing following experimental SCI. Throughout the review, we provide information on the correlation between existing experimental and clinically used autonomic tests. Finally, the review contains a comprehensive set of tables and illustrations to guide the reader through the complexity of autonomic assessment and dysfunctions observed following SCI. Conclusions: A wide variety of techniques exist to evaluate autonomic function in experimental animals with SCI. The incorporation of autonomic assessment as outcome measures in experiments testing treatments or interventions for SCI should be considered a high, clinically relevant priority. Spinal Cord (2009) 47, 2–35; doi:10.1038/sc.2008.61; published online 10 June 2008

Keywords: spinal cord injury; cardiovascular; respiratory; bladder; bowel; sexual

Introduction Although it is generally appreciated that spinal cord injury nervous system. Despite the widespread effects of SCI on (SCI) disrupts all types of communication between the brain autonomic control, it is only recently that autonomic and periphery below the lesion, the outcome of SCI is still function following SCI has received significant attention in commonly described in terms of motor function. The clinical research.4–6 The delay in addressing the autonomic pervasive mental association between SCI and paralysis is effects of SCI has not only limited their appreciation among reflected in recent headlines announcing a study on basic scientists and clinicians, but also efforts to develop new experimental SCI published in Nature Medicine.1 The treatments or rehabilitation strategies targeting autonomic headlines read ‘Scientists move toward helping paralysis function following SCI. These shortcomings are not insig- patients’2 and ‘paralysis cure’.3 nificant, as autonomic dysfunctions represent the primary In keeping with this perspective, an overwhelming causes of morbidity and mortality following SCI.7,8 In number of clinical studies have focused on the effects of addition, individuals with SCI have identified recovery of SCI on voluntary movement and the role of the somatic autonomic functions as a high priority for improving their quality of life.9 Recent data demonstrate that autonomic Correspondence: Dr AV Krassioukov, International Collaboration on Repair function is not reliably predicted by the degree of residual 10,11 Discoveries, University of British Columbia, 2469-6270 University Blvd., motor or sensory function. Together these results suggest Vancouver, British Columbia, Canada V6T 1Z4. that there is an imbalance between clinical priorities and the E-mail: [email protected] general focus of SCI research. 4These authors contributed equally to this work. Received 20 February 2008; revised 5 May 2008; accepted 5 May 2008; A similar imbalance exists in animal research. This is published online 10 June 2008 highlighted by a systematic review of the animal SCI Autonomic assessment in experimental SCI JA Inskip et al 3 literature (Figure 1; see ‘Methods’). We reviewed published tional definitions of autonomic dysfunctions that are studies of animals with experimental SCI, and identified present in animals following SCI. Finally, there may be a studies with functional outcomes designed to evaluate lack of agreement on (and awareness of) well-designed, motor, sensory or autonomic function. While the total clinically relevant tests that have been validated to evaluate number of studies increased dramatically over the past 27 autonomic functions in animals with SCI. years, published studies with motor outcomes dramatically The main goal of this review is to provide the scientific outnumber those with sensory or autonomic outcomes. The community with an overview of the current methods used to disproportionate number of studies focusing on motor assess the autonomic function of animals with SCI. For six recovery after experimental SCI represents a significant aspects of autonomic functionFcardiovascular function, mismatch between the clinical priorities of improved respiratory function, GI function, lower urinary tract (LUT) autonomic function and the direction of SCI research in function, sexual function and thermoregulationFwe review animals. the innervation of the system in humans and rats, the When the functional autonomic outcomes are separated clinical implications of dysfunction following SCI and the according to organ system dysfunction, it is immediately tests or techniques that are currently available to evaluate apparent that not all aspects of SCI-induced autonomic function after experimental SCI. We also include functional dysfunction have been examined to the same extent. The tests that have been developed in other animal models, but available data characterize mainly bladder and cardiovascu- that appear to be applicable to animals with SCI. Each test is lar functions in SCI animals, whereas sexual function, reviewed in terms of its methodology, the type of informa- gastrointestinal (GI) function and thermoregulation remain tion that it provides and the available data in animals with essentially uninvestigated. These findings are particularly SCI. We hope to improve the understanding of functional alarming, as recovery of sexual function in particular has tests used to evaluate autonomic function and to increase been identified by both paraplegics and quadriplegics as an their incorporation in SCI experiments, particularly those urgent priority.9 studies testing potential therapeutic agents (see Table 9). We There are several likely reasons for the paucity of research also hope that this review will be useful to veterinarians in addressing autonomic dysfunctions following SCI. The clinical practice, to add to their arsenal of assessments of complex organization of the , animals with naturally occurring SCI. Throughout the review and its involvement in the control of almost every system in we also highlight clinical assessments that are similar to the the body, makes it difficult to select appropriate functional experimental methods we describe. The use of similar tests in tests. There is also some confusion surrounding the opera- the clinic and the laboratory is valuable because it facilitates

Figure 1 Autonomic dysfunction remains underrepresented in experimental spinal cord injury (SCI). (a) The total number of publications reporting the functional outcome in animal models of SCI has increased steadily since 1980. However, the rate of increase is dramatically different between studies characterizing function/dysfunction of different divisions of the nervous system. At every time period examined, published studies with motor outcomes far outnumber published studies investigating autonomic or sensory function. The disparity is particularly pronounced in the past 7 years, when studies incorporating a motor outcome outnumber publications with any autonomic outcome by more than four times. (b) When published data on autonomic function are categorized according to organ system dysfunction after SCI, it is clear that not every area is equally represented. Lower urinary tract (LUT) function/dysfunction is the best-characterized component of experimental SCI, whereas experiments studying sexual, gastrointestinal and thermoregulatory function remain comparatively scant. However, when we compare the number of published studies characterizing motor outcome in the past 7 years (554) with the number of published studies characterizing LUT function in the same period (60), it is obvious that every aspect of SCI-related autonomic dysfunction should be considered a priority in animal research.

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Table 1 Search terms used to identify studies of motor, sensory and autonomic outcome of experimental SCI

Motor Sensory Autonomic Autonomic; sympathetic; parasympathetic

Lower urinary tract Cardiovascular Respiratory Sexual Gastrointestinal Thermoregulation

Motor Sensory Urinary Cardiovascular Respiratory Sexual Gastrointestinal Thermoregulation Movement Sensation Bladder Blood pressure Breathing Uterus Bowel Sweating Walking Touch Micturition Lung Vagina Colon Piloerection Stepping Proprioception Renal Hypotension Menstruation Locomotor Pain Dysreflexia Menstrual Locomotion Allodynia Heart Penis Postural Hyperalgesia Arrhythmia Erection Spasticity Dysaesthesia Ejaculation Paresthesia

Each search term was used in combination with ‘spinal cord injury’. Searches were limited to animal studies published in English within specified dateranges(Figure1). Abstracts and manuscripts were reviewed to obtain the final publication count in each area: see Methods for details and Table 2 for inclusion/exclusion criteria.

the translation of our knowledge about autonomic function Table 2 Criteria used to identify studies with relevant functional and dysfunction following SCI between the bench and the assessment after experimental SCI bedside. Included Excluded

All experimental models of SCI K Clinical (veterinary) SCI Methods: literature review (for example, traumatic, K In vitro and ex vivo preparations ischemic) in all species K Review articles A systematic literature review was conducted using PubMed to identify studies that incorporated functional assessment Studies incorporating functional K Studies detected by the search assessments of SCI, including criteria that contained no after experimental SCI. Each search was limited to animal those that: functional assessments (For studies published in English, and to specified publication K Characterize an outcome example, motor neuron atrophy date ranges. Search terms were always used in combination measure after SCI) with ‘spinal cord injury’. A separate search was performed K Characterize function/ K Studies that only monitored dysfunction vital signs during surgery or using each of the terms listed in Table 1. The abstracts K Use functional outcome to treatment returned by each search were reviewed to identify studies assess manipulation or K Electrophysiological studies of with relevant functional outcomes; inclusion and exclusion treatment individual neuron properties criteria are listed in Table 2. If the outcome measure(s) used (versus neurological function) K Studies of spinal cord blood in the study were not evident from reading the abstract, the flow/lesion perfusion or ‘Methods’ section of the paper was reviewed. microperfusion (versus systemic Publications were not counted twice in the same section cardiovascular function) (that is, no paper was counted as both locomotor and K Studies testing the effects of vasopressor therapy as a movement), but some were considered as spanning two treatment for SCI sections (that is, some papers contained both motor and sensory functional assessments). All studies incorporating Abstracts returned by literature searches were reviewed; if necessary, manu- the functional outcome of interest were included, regardless scripts were also reviewed to determine what outcome measures were included in the study. Studies were included/excluded according to the listed criteria. of the objective of the study. For example, studies aimed at characterizing the effects of a treatment on pain after SCI that also included motor testing were counted as both motor innervation of the cardiovascular system has important and sensory. Finally, publications were categorized according ramifications for the pattern of cardiovascular dysfunction to the intent of the outcome measure. For example, if that emerges after SCI. Here we review the most relevant authors used a ladder-walking test to assess locomotor features of cardiovascular innervation (Figure 2), the details 12,13 function, the study was counted as motor (even though of which have been extensively reviewed elsewhere. there are presumably proprioceptive components to the performance). Clinical implications of cardiovascular dysfunction following SCI Unlike dysfunction of the LUT, which can be described in Cardiovascular function general terms for suprasacral SCI, cardiovascular dysfunction varies dramatically with level of injury,14,15 its severity Autonomic innervation of the cardiovascular system determined by the relative loss of supraspinal control over In humans and animals with an intact neuraxis, both tonic spinal sympathetic outflow.16 Cervical SCI disrupts suprasp- neurogenic and reflex autonomic control of the cardiovas- inal connections to preganglionic sympathetic innervation cular system ensure adequate regional blood supply under a of the heart and blood vessels, eliminating tonic excitatory wide range of physiological conditions. The autonomic input to these organs. If the injury occurs at T5 or above,

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Figure 2 Autonomic innervation of the cardiovascular system. The major organs of the cardiovascular system are the heart and the blood vessels. The heart receives both parasympathetic and sympathetic innervation. Parasympathetic efferents travel to the heart in the vagus nerve, which exits the (CNS) at the level of the medulla. The vagus nerve innervates the atria, nodes and Purkinje fibers via local cardiac ganglia, and vagal activity decreases heart rate, contractility and conduction velocity. Sympathetic activity has an opposite, stimulatory effect on the heart. All tissues of the heart receive sympathetic input from the upper thoracic (T1–T5) cord. Blood vessels are under sympathetic control, and vessels supplying the splanchnic regionFthe liver, spleen and intestinesFare most important in cardiovascular control. The splanchnic bed is densely innervated, highly compliant and contains approximately one-fourth of the total blood volume in humans at rest.403 As such, it is the primary capacitance bed in the body. Sympathetic outflow to the splanchnic bed exits the thoracolumbar cord (T5–L2) and provides tonic . The relative amount of sympathetic and parasympathetic activity governing cardiovascular control is determined (in part) by information from two types of afferents: and chemoreceptors. Baroreceptors in the aortic arch, carotid sinus and coronary arteries detect changes in arterial pressure, and chemoreceptors in the carotid bodies respond to changes in partial pressures of oxygen and carbon dioxide in the blood. activity is the primary drive for rapid blood pressure adjustment. Baroreceptor afferents travel primarily in the vagus nerve and the glossopharyngeal nerve to reach the medulla. Abbreviations: AR, adrenergic receptors; CVLM, caudal ventrolateral medulla; DMNX, dorsal vagal motor nerve; g, ganglion; mAChR, muscarinic cholinergic receptors; NA, nucleus ambiguous; n, nerve; NTS, nucleus of the solitary tract; P2X, purinergic receptors; RVLM, rostral ventrolateral medulla; ( þ ) denotes excitatory synapses; (À) denotes inhibitory synapses. supraspinal control over sympathetic innervation of the heart rate and resting blood pressure.21 Cervical SCI also splanchnic vascular bed is lost, jeopardizing blood pressure abolishes circadian blood pressure rhythms 22–24 and blunts control. For this reason, injuries at and above T5–T6 are most the cardiovascular responses to exercise.25 In addition to low likely to precipitate severe cardiovascular dysfunction.14,17 resting blood pressure, many people with high SCI experi- The acute phase of clinical SCI is marked by neurogenic ence (OH), a decrease in blood , a period of profoundly altered cardiovascular con- pressure that occurs with assumption of a sitting position.26 trol.18 This phenomenon is particularly pronounced follow- These individuals are also prone to autonomic dysreflexia ing cervical SCI, which frequently induces severe (AD), episodes of paroxysmal hypertension, often accom- hypotension and .14,17 After panied by baroreflex-mediated bradycardia, induced by resolves, basal cardiovascular parameters remain altered in sensory stimulation below the level of the injury.17 As a people with cervical and high-thoracic SCI.19 People with group, cardiovascular disorders are the most common mid-thoracic SCI typically have elevated heart rates,20 underlying or contributing causes of death in people whereas people with higher injuries often present with low with SCI.8,27

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Assessment of cardiovascular function following experimental SCI with the level and severity of injury, as well as the SCI alters the connectivity of autonomic circuits between the experimental conditions, the majority of experiments are brainstem and spinal cord that governs cardiovascular conducted at least 2 weeks after SCI when AD is pronounced. control and modifies sympathetic intraspinal and neurovas- This well-characterized model has been used extensively to cular transmission resulting in altered vascular smooth identify mechanisms contributing to the development of muscle responses.28–34 These phenomena (as well as others) AD; still, only a few studies have examined the severity of AD are likely to contribute to SCI-induced cardiovascular as an outcome measure when testing therapies for SCI dysfunction. Here we focus on the systemic cardiovascular (Table 9). outcome of experimental SCI. Several aspects of SCI-induced Compared to its hypertensive counterpart, OH has cardiovascular dysfunction have been successfully modeled received little attention in experimental SCI. Some clues to in experimental animals. Cardiovascular parameters of mechanisms of OH are provided by animal models of animals with SCI are monitored from direct arterial blood microgravity-induced cardiovascular deconditioning, in pressure measurements: indirect measurements of blood which microgravity is simulated by hindlimb unloading pressure (collected using a tail cuff, for example) have never (HU).75–77 Animals exposed to HU experience a pronounced been used in animals with SCI. Arterial blood pressure is drop in blood pressure when subjected to head-up tilt.78 taken either directly from a fluid-filled cannula or via Considering this, we are currently developing an animal radiotelemetric monitoring of animals with chronically model of OH after SCI. We have recently observed OH in rats implanted cannulae. with complete transection injury at T3–T4 (JAI, LMR and AVK, unpublished observations). When these unanesthe- tized rats are subjected to passive head-up tilt 1 month after Short-term cardiovascular assessment using fluid-filled cannulae injury, they experience a pronounced drop in blood pressure The bulk of available cardiovascular data from animals with (Figure 3); uninjured rats subjected to the same maneuver SCI has been collected using arterial cannulae implanted exhibit either a rise in blood pressure or very little change. acutely prior to data collection. Typically, the cannula is Blood pressure data collected directly from arterial cannu- placed in one carotid or femoral artery hours or days before lae have also been used to monitor animals in the early data collection, although it is possible to maintain cannulae phases of recovery from SCI. Neurogenic shock is generally for at least 1 week with daily . Cannulae are filled considered to be less pronounced in experimental animals with heparin solution to preserve patency and tunneled than in human SCI. This notion might stem from the fact subcutaneously for externalization. After arterial cannulae that the overwhelming majority of experiments are per- are implanted, animals are housed singly to protect cannulae formed in animals with thoracic SCI. Animals with cervical from cage-mate chewing. Cardiovascular parameters can be SCI may experience neurogenic shock that is more compar- monitored directly in conscious animals in their home cage able to the clinical situation. Rats with complete transection by connecting the cannulae to a pressure transducer: this between C7 and T1 developed both hypotension and method has been used frequently to study many types of bradycardia.79 Mean arterial pressure fell precipitously (by cardiovascular dysfunction in animals with SCI. approximately 30 mm Hg) in the first 24 h after injury, and The aspect of cardiovascular dysfunction that has been slowly recovered to preinjury levels (around 100 mm Hg) by best characterized in rodent models of SCI is AD, episodic 9 days after injury. Heart rate also decreased dramatically hypertension that has been described in both rats and mice, (from about 360 b.p.m. to about 300 b.p.m.) and remained and is induced by visceral and somatic stimuli (see Table 3 significantly lower than preinjury heart rate for 9 days (the for detailed references). The nature of rodent AD and its duration of the study). Owing to the challenges involved in inciting stimuli is very similar to that described in humans. animal care, there are very few data on animals with high, Rats, like humans, typically experience AD when SCI occurs severe SCI. These animal models are currently being devel- at or above T5–T6. In one study, bladder filling induced AD oped (for example Pearse et al.80) and should facilitate in rats with severe contusion SCI at T4, but not in rats with our understanding of cardiovascular control following severe T10 contusion.68 The most common experimental cervical SCI. stimulus for AD is colorectal distension (CRD), in which a Cardiovascular data collected from animals with acutely balloon catheter is inserted into the colon through the anus implanted arterial cannulae have also been subjected to in the conscious animal.28 Air is infused into the balloon to spectral analysis. This type of analysis exploits the relation- mimic the pressure of several fecal boli in the colon. ship between frequency of spontaneous fluctuations in Distension is typically maintained for 1 min, and arterial cardiovascular parameters and the activity of systems (local, pressure is monitored before, during and after CRD. sympathetic and parasympathetic) governing cardiovascular Formerly considered to be associated with chronic SCI, AD control. Spectral analysis has been validated in humans,81,82 may also emerge during the acute stages in both animals and dogs82–84 and rats,85 and provides indices of autonomic humans.74 Rats with complete transection of the spinal cord nervous system activity from relatively short segments of at T5 develop AD in response to CRD as early as 24 h after continuous recordings of blood pressure (and in humans, injury.28 The severity of AD subsequently decreases during electrocardiogram signals). Spectral analysis has been ap- the first post-injury week, and AD is relatively mild at 7 days plied to examine blood pressure variation in rats with acute following T5 SCI.28,38 Thereafter, the severity of AD in- SCI.70 At 1 and 6 days after complete transection at T4–T5, creases. Although the actual rise in blood pressure varies rats exhibited reduced power in the low-frequency range,

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Table 3 Overview of studies characterizing or targeting cardiovascular dysfunction after experimental SCI

Technique used Species Injury model Time range post-injury References

Acute arterial cannulation Basal cardiovascular parameters Rat T1–T4 clip compression o4h 35–37 1.5 months 38 C6/7 Tx; T2 Tx; T9 Tx 4 h 38 p1 week 39 T4–T7 Tx 0 h–6 weeks 40 p1 week 41 T5–T8 contusion o1h 42–44 T9 clip compression 1, 2 h 45 Rat, cat C7 Tx (ligature crush) o1h 46 Cat T6 contusion p24 h 47 Dog C4 Tx o1h 48 Monkey T5 balloon compression 49 Autonomic dysreflexia Colorectal distension Mouse T2 clip compression 2 weeks 50 T2 Tx 2 weeks 50–52 Rat C7–T1; C8 Tx 3 days 53,54 2–3 months 55 T4–T5 clip compression p1 week 38,56 2–4 weeks 38,57–59 4–6 weeks 38,59 T3–T5 Tx p24 h 40,60,61 p1 week 28,40,60 2–4 weeks 28,34,40,60–65 2–3 months 55 Bladder distension Rat C7–T1 Tx 24 h 66 p1 week 67 T4 contusion 4 weeks 68 T5 Tx p1 week 28 Somatic stim. Mouse T2 clip compression 2 weeks 50 T2 Tx 50–52 Rat T4 clip compression 2–6 weeks 59 Vag.-cerv. stim. Rat T7 Tx 1–3 weeks 69 Spectral analysis Rat T1–T5 Tx p1 week 70,71 Telemetric monitoring Rat C7–T1 Tx 2–3 months 55 T4–5 Tx p24 h, p1 week 40 4–6 weeks 40,72 2–3 months 73 T4 clip compression 1.5 months 38

Abbreviations: stim., stimulation; Tx, transection; vag.-cerv., vaginocervical. Studies have been classified by the technique used to assess cardiovascular function. Studies employing a combination of techniques are referenced under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together. indicative of reduced sympathetic tone (or increased para- of data collection, as well as possible effects of recent surgery sympathetic tone85) that affects blood pressure control. for cannula implantation, may contribute to stress in the There are currently no data applying spectral analysis to animals during data collection. more chronic experimental SCI. However, as this analysis is related to clinical measures of autonomic control following human SCI,86 it represents a readily translatable tool for autonomic assessment in experimental animals and humans Telemetric monitoring of cardiovascular parameters with SCI. Telemetric monitoring provides continuous physiological information about the cardiovascular function of conscious, freely moving animals over a long time course. This Advantages/disadvantages. Blood pressure measurements technique was first applied to animals with SCI in our collected directly from fluid-filled cannulae provide high- laboratory (Figure 4),38 and allowed us to characterize resolution cardiovascular data with a relatively modest changes in resting blood pressure and heart rate in rats investment in equipment. However, preserving catheter during recovery from high-thoracic SCI. Rats were instru- patency for repeated measurements in the same animals mented with a telemetric transducer (catheter implanted can present a challenge. For this reason, this method into the descending aorta) 1 month prior to SCI, and arterial typically only provides a snapshot of information about pressure data were transmitted as a radiofrequency signal to a cardiovascular function in each animal (that is, taken at a receiver under the cage. The recovery time after transducer single time point). The presence of investigators at the time implantation was critical, as the effects of simultaneous

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Figure 3 Representative tracings using fluid-filled cannulae to assess blood pressure during head-up tilt. One month after complete T3–T4 transection injury, conscious rats with a left carotid artery cannulation subjected to a passive 901 head-up tilt experienced a pronounced drop in blood pressure. Uninjured rats subjected to the same maneuver exhibited either a rise in blood pressure or very little change (JAI, LMR and AVK, unpublished observations).

transducer implantation and SCI can impede blood supply to catheter maintenance, or restraint for catheter connection, the hindlimbs.38 telemetry can be considered the gold standard for cardio- Telemetric monitoring of animals with SCI suggests that vascular assessment in conscious experimental animals. It SCI-induced alterations in basal cardiovascular control are does not eliminate stress due to handling when animals similar between rats and humans. Rats with T5 clip must be loosely restrained for cardiovascular measurements compression SCI experience transient hypotension and (such as during CRD to induce AD), so this remains a persistent following injury.38,87 Telemetric mon- consideration in telemetric studies. Most significantly, itoring revealed that blood pressure recovered to preinjury adopting telemetric monitoring entails considerable invest- levels by 48 h after SCI, and remained equivalent to preinjury ment in equipment. Perhaps for this reason, data in animals levels for at least 6 weeks following injury. Heart rate was with SCI remain scant (Table 3). increased relative to preinjury levels by 3 days after SCI, and remained elevated for at least 6 weeks. Diurnal fluctuations in blood pressure and heart rate returned approximately 5 Respiratory function days after injury.38 Interestingly, recent telemetric data demonstrate that rats with more rostral injuries (complete Innervation of the respiratory system T4 transection) experience persistent hypotension and Coordinated activity of somatic (diaphragm and accessory tachycardia, with altered basal blood pressure and heart rate respiratory skeletal muscles) and autonomic (smooth mus- for at least 6 weeks following SCI.40 Thus, it seems that in cles of the bronchial tree) nervous systems is crucial for rats, as in humans, severity of cardiovascular dysfunction normal respiration. Here we review efferent and afferent varies with both level and severity of SCI. Radiotelemetry has innervation as it pertains to respiratory dysfunction that also been used to investigate AD induced by CRD, which is emerges following SCI (Figure 5). For simplicity, we omit similar in progression between rats with T5 clip compression many important aspects, and the interested reader is directed and rats with T4 complete transection (Figure 4).38,40 to comprehensive reviews on the subject (for example, see Brading12 and Canning and Fischer88).

Advantages/disadvantages. As data collection is continuous, Clinical implications of respiratory dysfunction following SCI telemetric monitoring is extremely informative. Since it Respiratory dysfunction after SCI is determined by relative avoids the potential stressors of intra-arterial catheterization, loss of descending autonomic innervation to the respiratory

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Figure 4 Use of telemetric monitoring to assess basal cardiovascular parameters (a, b) and autonomic dysreflexia (c, d) after experimental spinal cord injury (SCI). Rats were instrumented with radiotelemetric pressure transducers and cannulae were implanted into the descending aorta 1 month prior to T5 clip compression SCI. (a, b) Telemetric monitoring permitted data collection in freely moving rats for 3 days prior to SCI (to establish reliable uninjured baseline responses) and for the first 10 days following SCI. Mean values (±s.e.m.) of 3 h intervals for five rats are shown, and light and dark periods are indicated on the x axes. (c, d) Autonomic dysreflexia (AD) was examined at 7 and 28 days following SCI. Average responses (±s.e.m.) to colorectal distension in five rats are shown. This figure is reprinted from Mayorov et al.38 muscles. Injuries above C3 paralyze the diaphragm, and This injury reliably disrupts innervation to one-half of the ventilatory support is typically required to sustain life. After diaphragm. Recently, cervical hemicontusion models have C3–C5 SCI, the diaphragm is partially denervated, and been developed that also induce respiratory deficits due to inspiration is compromised; still, most individuals with hemidiaphragm paralysis.98,99 Data from both injury models C4–C5 SCI do not require artificial ventilation (or can be have increased our understanding of endogenous plastic weaned during recovery89). In lower cervical SCI, innerva- processes that might improve respiratory function following tion of the primary (and some accessory) inspiratory muscles SCI (Table 4; reviewed in Zimmer et al.134). A few studies have is preserved, but ventilation is still impaired, because also examined the potential for regenerative therapies to denervated intercostal muscles do not coordinate chest restore diaphragm function (Table 9). The obvious limitation expansion with diaphragm descent.90 Finally, any SCI above of these models is that they are incomplete injuries, and thus L1–L2 denervates abdominal muscles, reducing the effec- do not model all aspects of clinical SCI. As more models of tiveness of coughing. severe cervical SCI are developed, our understanding of The clinical ramifications of respiratory dysfunctions respiratory dysfunction after SCI is likely to improve. following SCI are severe, and encompass pneumonia, Fortunately, outcome measures used to assess respiratory atelectasis, bronchitis, reduced lung volumes and compli- function are similar between experimental and clinical SCI, ance, sleep apnea and respiratory insufficiency or dyspnea, facilitating translation of these data. particularly during exercise.91–95 The incidence and severity of respiratory dysfunction increases with the level of SCI. Respiratory complications are the leading cause of death in acute SCI.96 In chronic SCI, respiratory dysfunction con- Phrenic nerve conduction tributes significantly to mortality,8 and is associated with Phrenic nerve conduction testing is used clinically for reduced quality of life.97 planning phrenic nerve or diaphragmatic pacing for assisted activation of the diaphragm.135–137 In animals, phrenic nerve responses during spontaneous breathing or spinal Assessment of respiratory function following experimental SCI cord stimulation are examined in anesthetized, paralyzed The most common experimental model used to study SCI- (mechanically ventilated) animals.101–103,121,122 These data induced respiratory dysfunction is rat cervical hemisection. have been used extensively to characterize respiratory

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Figure 5 Innervation of the respiratory system. The main respiratory muscles are the diaphragm, intercostals and abdominals. The diaphragm is the major inspiratory muscle and is innervated by phrenic motor neurons that lie in the cervical spinal cord (C3–C5 in humans; C3–C6 in rats404). Innervation of respiratory intercostal and abdominal muscles exits the thoracolumbar spinal cord, from T1–T11 and T7–L2, respectively. Activity of these muscles (as well as that of accessory muscles) is modulated by autonomic premotor neurons in the VLM, which project to motor neurons in the spinal cord. The airways receive both parasympathetic and sympathetic inputs. The parasympathetic nervous system provides the most important innervation to the smooth muscle of the airways, and is thus most important in controlling its diameter. Preganglionic parasympathetic neurons originate in the NA, and innervate the trachea and the bronchi via the laryngeal and vagus nerves (respectively). Parasympathetic innervation is predominately cholinergic and its action is excitatory, reducing airway diameter (via mAChR). Sympathetic innervation of smooth muscle is comparatively scant. Preganglionic sympathetic axons exit at T4 and travel to paravertebral ganglia, and post-ganglionic adrenergic fibers elicit bronchodilation, acting through b-AR. The airways also have extensive afferent innervation. The most important afferents regulating respiration are vagal Ad, with cell bodies in the nodose ganglia and central axons projecting to the NTS. Abbreviations:Ad, mechanoreceptors; b-AR, b-adrenergic receptors; C, cervical spinal cord; g, ganglion; L, lumbar spinal cord; mAChR, muscarinic cholinergic receptors; NA, nucleus ambiguous; nAChR, nicotinic cholinergic receptors; NE, ; n, nerve; NTS, nucleus of the solitary tract; T, thoracic spinal cord; VLM, ventrolateral medulla; ( þ ) denotes excitatory synapses; (À) denotes inhibitory synapses.

plasticity after SCI, particularly during the crossed phrenic latent pathway to restore diaphragmatic function. This phenomenon (CPP).134,138 pathway also becomes spontaneously active over time The CPP relies on a functionally latent bulbospinal following SCI, but this spontaneous return of activity may pathway that innervates the diaphragm bilaterally. When not be sufficient to restore diaphragmatic function.104 A the hemidiaphragm is paralyzed by C2 hemisection, hypoxia series of phrenic nerve function studies has revealed induced by contralateral phrenic nerve section activates the that systemic delivery of xanthines (adenosine receptor

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Table 4 Overview of studies characterizing or targeting respiratory dysfunction after experimental SCI

Technique used Species Injury model Time range post-injury References

Phrenic nerve conduction Rat C2 hemicontusion 1 week 99 4–6 weeks 99,100 C2 hemisection p24 h 101–118 p1 week 104,116,117,119,120 2–4 weeks 103,119,121–124 4–8 weeks 121,125 2–4 months 116,117,126 C4/5 contusion 2–11 weeks 100

Pneumotachometry Rat C2 hemisection 1–2 months 127,128 Dog T2, T4, T8 (seg. epid.) Immediate 129 Turtle D8–D10 Tx 4, 8 weeks 130

Diaphragm EMG Rat C2 hemisection p24 h 105,117,131 p1 week 119 2–4 weeks 119 90 days 117 C2 hemicontusion 7 days, 1 month 99 Dog T2, T4, T8 (seg. epid.) Immediate 129 Mouse C2 hemisection 1–48 h 132

Plethysmography Rat C2 hemisection 2, 3, 5 weeks 122 C4/5 hemicontusion 24 h, 1, 2, 4, 6 weeks 98 T8 contusion 24 h, 7 days 133

Abbreviations: EMG, electromyography; seg. epi., segmental epidural; Tx, transection. Studies have been classified by the technique used to assess respiratory function. Studies employing a combination of techniques are referenced under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together. antagonists) can activate this pathway without phrenic Pneumotachography was also applied to verify the func- nerve section, to restore respiratory drive to phrenic motor tional significance of crossed phrenic pathways.128 neurons following SCI.105–107,123,126 These data, and pre- liminary clinical experience, suggest that xanthine treat- ment may represent a viable therapeutic option in weaning Diaphragmatic electromyography individuals with SCI from ventilatory support.139 Similar to clinical practice, electromyography (EMG) of the Phrenic nerve recording has also been used to test the diaphragm can be performed in conjunction with phrenic efficacy of olfactory ensheathing cell (OEC) transplantation nerve conduction studies in animals with SCI. For example, in improving the respiratory outcome of experimental rats that received OECs at the time of C2 hemisection SCI140,141 (Table 9). In these experiments, rats with high recovered both ipsilateral phrenic nerve activity and ipsilat- cervical hemisection (that abolished ipsilateral phrenic nerve eral diaphragm activity 3–6 months after treatment and activity) received OEC transplantation at the time and site of injury.141 A recent study used diaphragmatic EMG to test the injury. In one study, rats that received OECs recovered effect of administering a N-methyl-D-aspartic acid (NMDA) spontaneous respiratory rhythm in the ipsilateral phrenic receptor antagonist (MK-801) in acute cervical SCI.131 In nerve by 2 months after SCI.140 In another set of experi- these experiments, i.p. MK-801 administration after C2 ments, ipsilateral phrenic nerve activity recovered to hemisection was associated with both recovery of ipsilateral approximately 80% of contralateral nerve activity by 3–6 diaphragm function and upregulation of NMDA receptor months post-SCI.141 In the latter set of experiments, the subunit NR2A. authors transected the contralateral spinal cord to demon- strate that a significant proportion of this recovery was due to ipsilateral projections. However, the underlying mechan- ism of recovery is not known. Advantages/disadvantages (phrenic nerve conduction, pneumota- chometry and diaphragmatic EMG). Although diaphragmatic EMG, phrenic nerve recordings and pneumotachometric Pneumotachometry evaluations have clinical correlates, they are much more Pneumotachometry is the evaluation of the respiratory invasive procedures in the experimental laboratory than in volumes and rate that can be readily applied in conjunction the clinical setting. These experiments are technically with phrenic nerve recording. This type of assessment was demanding and terminal preparations. Although they important in identifying altered breathing patterns in rats provide a quantitative and informative index of diaphrag- with unilateral cervical SCI.127 Rats with C2 hemisection matic function, other outcome measures may be more exhibit a reduced expiratory volume and an increased suitable when respiratory function is not the sole or primary respiratory rate to preserve total (minute) ventilation.127 focus of an experiment.

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Plethysmography prolonged orocecal transit time (OCTT) following An alternative outcome measure for assessing respiratory SCI.154–157 The most common complications are lower GI function in experimental SCI is whole-body plethysmogra- tract dysfunctions and most research has focused on their phy (WBP), in which respiratory function is determined in identification and resolution. These typically present as conscious animals, without invasive instrumentation. Prior constipation, compaction and fecal incontinence, and can to experimentation, rats are typically trained to become trigger both physical and psychological problems that acclimated to the plethysmography chamber to prevent restrict lifestyle choices and disrupt rehabilitation and confounds of stress. In a recent study, WBP was used to overall quality of life of individuals with SCI.158,159 characterize respiratory function in rats with a C5 hemi- The term ‘neurogenic bowel’ describes the loss of neuronal contusion SCI.98 These rats exhibited respiratory deficits that control to the colon, and resulting dysfunction.160 The are reminiscent of clinical SCI:142 specifically, their ability to neurogenic bowel can be divided into two main types, each augment tidal volume in response to hypercapnic challenge with characteristic colonic dysfunctions; supraconal lesions was impaired 4 weeks after severe SCI. This technique has result in damage to descending supraspinal pathways (upper also been used to verify the altered breathing pattern motor neuron bowel syndrome) and infraconal (cauda induced by C2 hemisection previously reported in anesthe- equina or pelvic nerve lesions) damage to motor and tized rats.122 Although spirometry is more commonly used in parasympathetic innervation to the colon (lower motor the clinic, WBP has recently been validated for respiratory neuron bowel syndrome).160 The upper motor neuron evaluation of people with SCI.143,144 bowel, or hyperreflexive bowel, is associated with spastic activity in the colon and external anal sphincter (EAS), Advantages/disadvantages. The most obvious advantage of which interferes with the voluntary ability to defecate but F WBP is that it is not a terminal experiment. Rather, leaves the bowel reflex intact the basis for bowel emptying respiratory data can be collected in the same animals at using chemical or mechanical stimuli. The lower motor different time points following SCI. It does not provide a neuron, or areflexive, bowel is associated with a relaxed direct index of diaphragm function, but does provide colon, perturbed peristalsis, slow stool propulsion and 160 clinically relevant indices of respiratory function in experi- constipation. mental animals. As it also requires less technical expertise than other methods, WBP may represent an attractive Assessment of gastrointestinal function following experimental method for many laboratories investigating respiratory SCI dysfunction after experimental SCI. Given that SCI primarily affects GI motor control, the majority of GI assessments following SCI consist of tests of Gastrointestinal function functional motility or contractile activity. This section focuses on functional GI assessments used in experimental Innervation of the gastrointestinal system animal models (Table 5), some of which have been used as The GI system control involves a complex interaction outcome measures for assessing autonomic function of the between the somatic nervous system (anal sphincters), both GI tract in humans following SCI and for testing clinically divisions of the autonomic nervous system, and the unique relevant therapies for restoring GI function. intrinsic enteric nervous system (ENS). The ENS controls the secretion, motility, blood flow, storage and evacuation of the Gastrointestinal transit with oral markers GI tract, and its basic organization and function is similar GI transit can be assessed in experimental animals by across species. The autonomic nervous system modulates the segmental dye recovery along the GI tract following oral intrinsic activity of the ENS, and is especially important at marker delivery.170,171 In the experimental animal, a non- the proximal and distal ends of the GI tract (Figure 6). More toxic, nonabsorbable dye is administered by gavage feeding detailed information about the interactions between the to the stomach. After the desired time interval, animals are autonomic nervous system and ENS can be found in recent euthanized, clamps are secured between each GI segment 145–147 reviews. and marker recovery is detected by spectrophotometry to quantify the amount of dye in each segment, an indicator of Clinical impact of gastrointestinal dysfunctions following SCI movement through the GI tract.170 Although the ENS does have some intrinsic functional Dye recovery has been used as an outcome measure in rats capacity, this is significantly impaired when it loses central with SCI at various levels.161,172 Rats with cervical or thoracic coordination following SCI. The disruption of autonomic spinal cord transection showed increased dye recovery in the innervation to the GI tract is primarily revealed by abnormal stomach and decreased recovery in the small intestine motor function as opposed to changes in secretory or throughout the first week after injury, indicating decreased absorptive function.148 GI problems are prevalent in both GE and overall GI transit.161 However, after 10 days post-SCI, the acute and chronic periods following SCI, and are a there were no differences in dye recovery between the significant cause of rehospitalization and morbidity.149–153 stomach and intestine, indicating that there is a recovery of Although there is some controversy over the effect of SCI on GE and overall GI transit.164 Unlike humans, these rats also the upper GI tract, there is evidence of gastric dilation, showed concurrent spontaneous recovery of bowel func- delayed gastric emptying (GE), gastric ulceration and tion.172 A follow-up study revealed that large bowel empty-

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Figure 6 Innervation of the distal gastrointestinal (GI) tract. The ENS is composed of two main ganglionated plexuses that lie between the longitudinal and circular muscle layers (myenteric or Auerbach’s plexus) and within the submucosal layer of the GI tract (submucosal or Meissner’s plexus). The myenteric plexus is continuous throughout the entire length of the GI tract, and is primarily involved in the coordination of smooth muscle activity, whereas the submucosal plexus is present primarily in the small and large intestines, where it controls secretion and absorption.145 Autonomic innervation of the GI tract is required to modulate the intrinsic activity of the enteric nervous system (ENS). This modulation is especially important in the distal GI tract (illustrated here), where the ANS coordinates storage and evacuation by regulating colon motility and resting anal sphincter tone.405,406 Parasympathetic innervation of the distal colon and rectum originates in the sacral cord (S2–S4), whereas the upper GI tract (to the level of the splenic flexure) is innervated by the vagus nerve (not illustrated here). Preganglionic parasympathetic neurons synapse directly on Auerbach’s plexus that enhances smooth muscle activity (via mAChR). Sympathetic innervation is mainly postganglionic, and arises from paravertebral and prevertebral ganglia of the abdominal and pelvic cavitiesFceliac, superior and inferior mesenteric and pelvic ganglia. Sympathetic neurons in prevertebral ganglia inhibit muscle and secretory activity indirectly, by noradrenergic modulation of activity in both the Meissner and Auerbach’s plexuses. The IAS receives both sympathetic and parasympathetic innervation, whereas the EAS is innervated by somatic fibers traveling in the pudendal nerve (S2–S4 in humans, L6–S1 in rats). Afferent information from this area travels in both the pelvic and pudendal nerves. Abbreviations:Ad, C, mechanosensitive primary afferents; aAR, a-adrenergic receptors; DR g, dorsal root ganglion; EAS, external anal sphincter; g, ganglion; IAS, internal anal sphincter; IM g, inferior mesenteric ganglion; mAChR, muscarinic cholinergic receptors; n, nerve; nAChR, nicotinic cholinergic receptors; NO, nitric oxide; ( þ ) denotes excitatory synapses; (À) denotes inhibitory synapses.

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Table 5 Overview of studies characterizing or targeting gastrointestinal dysfunction after experimental SCI

Technique used Species Injury model Time range post-injury References

GI transit with oral markers Rat T4–T5 Tx 30 min–7 days 161 1day 162,163 1–30 days 164 C7–T1 Tx 30 min; 6 h; 1, 3, 7 days 161 Cat T4 hemostat clamp 1–2 weeks 165

Manometry Rat T4 Tx 1 day–2 weeks 166 Cat T4 hemostat clamp 1–2 weeks 165 Dog T10 Tx 2–6 weeks 167

EMG Rat T9–T10 Tx p24 h 168 2 days–6 weeks 168,169 T9–T10 contusion 2 days–6 weeks 169

Abbreviations: EMG, electromyography; GI, gastrointestinal; Tx, transection. Studies have been classified by the technique used to assess gastrointestinal function. Studies employing a combination of techniques are referenced under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.

ing prevented the development of delayed GE and GI transit tail.168,169 Temporary bipolar EMG electrodes are implanted following SCI.162 Although the effects of SCI on the upper GI in the EAS, with the external wire attached to the tail and tract are clinically still somewhat controversial, with some connected to a preamplifier. After recording baseline EAS groups reporting GE delays and others demonstrating EMG activity, EAS distension is initiated with a plastic probe normal GE,155–157,173 these results suggest that delayed GE (used to mimic fecal bolus), and the resultant EAS contrac- is in fact secondary to lower GI delayed motility.162 This tions are recorded.168 Anesthetics are not normally used in corroborates previous research showing that the GE reflex is this assessment, as they significantly attenuate EAS hyper- mediated by vagovagal reflexes and is not disrupted by reflexia. sympathetic denervation of the upper GI tract.174,175 This technique has also been used to assess functional Visualization techniques, similar to orally administered recovery of autonomic reflexes after the period of spinal radionucleotides used to assess GI motility clini- shock, and the development of hyperreactive autonomic cally,156,176,177 can also be applied in the experimental reflexes.169 EAS , reflected in prolonged burst setting. Video fluoroscopy has been recently used to assess duration of EAS activity, developed 2 days post-contusion at the functional benefits of colonic electrical stimulation as a T9–T10 and resolved to preoperative levels, not significantly treatment to improve colonic transit in rats178,179 and SCI different from controls, by 6 weeks post-injury. In contrast, cats.165 These results suggest that colonic transit times are spinally transected animals developed EAS hyperreflexia 7 improved with the use of colonic electrical stimulation.180 days post-injury and did not demonstrate any EAS reflex Magnetic resonance imaging (MRI) has also been used to recovery.169 This research demonstrates the sensitivity of EAS visualize GI motility by detection of solid food labeled with EMG recording and its potential as an objective assessment trace amounts of nontoxic iron oxide particles in rats.181 of pelvic autonomic reflexes. EMG recording from the jejunum has been recently adapted for use with telemetry, allowing for recording in Advantages/disadvantages. Oral marker delivery for evalua- awake and mobile rats.182 In this technique, an EMG tion of GI transit can require training and animal habitua- transmitter is implanted dorsally between the shoulder tion, particularly if gavage feeding is required. The detection blades and connected to the electrodes in the jejunum.182 of the markers can be performed as a terminal preparation, or by indirect visualization. The latter type of detection techniques (MRI or X ray) are attractive as they could be Advantages/disadvantages. EMG is technically demanding, easily combined with sensory or motor outcomes; however, usually terminal, and requires both training and specialized these techniques are more expensive and technically equipment. However, it represents the most sensitive and demanding. direct evaluation of the activity in the GI tract.

Electromyography Manometry EMG recording of EAS activity has been used as a tool to Manometry, the measurement of pressure changes within investigate the physiology and pathophysiology of the EAS different parts of the GI tract, has also been used to assess in rats with SCI.168 This preparation allows for the measure- colonic motility in rats following SCI.166 Similar to the ment of both baseline EMG activity in the EAS and the clinical technique183–185 a fluid-filled catheter is inserted contractions stimulated by EAS distension.168 Animals are into the colon and is attached to recording probes at restrained in a supine position using a loose-fitting cylinder, different regions along the colon; however, in the rat these or masking tape, to secure their torso, hind limbs and probes are secured with ligatures to allow for chronic

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 15 recording.166 To quantify motor activity, a motility index can Advantages/disadvantages. The hydrogen breath test is mini- be calculated that incorporates the amplitude, duration, mally invasive and could easily be used in conjunction with frequency and number of contractions. Using this techni- other assessments in SCI animals. It can be used at repeated que, spinally transected rats showed a reduction of distal intervals in the same animals. However, specialized equip- colonic motility acutely after injury, which returned to ment is necessary for detection of hydrogen in the breath. preoperative values after 7 days.186 Unfortunately, the combined motility index does not provide an indication of the functional motility in the colonFan important con- Urinary bladder function sideration given that one of the main problems following Innervation of the lower urinary tract SCI is the lack of coordinated peristalsis rather than lack of Although interspecies differences exist, the basic organiza- overall contractile activity.155 However, when used in tion and innervation of the LUT is similar among common combination with oral marker delivery, the effects of reduced 165,179,187 experimental species and humans (Figure 7). In general motility can be more clearly assessed. terms, storage of urine is sympathetically mediated, whereas micturition is elicited by parasympathetic activation: how- Strain gauge transducers ever, normal LUT function requires the coordinated activity of the sympathetic, parasympathetic and somatic nervous Strain gauge transducers have also been used to continuously systems. Here we provide only the basic scheme of LUT record gastric, small intestinal and colonic motility in awake rats.188 In contrast to manometry, this technique measures innervation, as neural control of the LUT and associated reflexes are comprehensively reviewed elsewhere.201–207 the pressure changes on the extraluminal surface of the GI tract.189–191 Although each transducer has only uniaxial sensitivity, by placing transducers at right angles to each Clinical implications of lower urinary tract dysfunction other in the same segment, both longitudinal and circular following SCI contractile muscle activities can be recordedFa good The manifestation of LUT dysfunction after SCI is 67,208 indicator of coordinated motility.189 This technique has similar in experimental animals, (Table 6) and 205,206,268–270 been used to model postoperative GI tract paresis,188 assess humans, and is broadly termed neurogenic neurochemical effects on GI motility192 and examine the bladder. The initial period following SCI is marked by functional effects of altered pacemaker activity in the gut.193 bladder areflexia and . When SCI occurs Although this technique has not yet been used in experi- at or below the sacral level (that is, infraconal; a lower mental SCI, its relative noninvasiveness and capacity for motoneuron injury) bladder areflexia persists. If the lesion chronic use makes it a promising way to measure functional extends rostrally to the thoracolumbar region to involve GI motility. sympathetic preganglionic neurons, the bladder neck may also become hypoactive. Suprasacral (supraconal) SCI (an upper motor neuron lesion) typically produces hyperreflexia Advantages/disadvantages (manometry and strain gauge of the smooth (detrusor) muscle of the bladder and tonic transducers). Both manometry and strain gauge techniques activation of the striated urethral sphincter. Therefore, require a fairly significant investment in training and sphincter contractions are dyssynergic with detrusor con- equipment. Manometry is ideal for acute measurements, tractions. Detrusor hyperreflexia and detrusor–sphincter but its use is restricted to the colon. The use of strain dyssynergia result from both a loss of tonic supraspinal gauges is surgically demanding, but does not interfere with inhibition and the emergence of aberrant spinal reflexes. The normal GI motility, and is therefore preferable for chronic clinical profile of LUT dysfunction is highly variable between recording.194 individuals with SCI, but can be generally described in terms of impaired continence (most common in supraconal SCI), impaired emptying (most severe in infraconal SCI) and Hydrogen breath test impaired sensation of bladder filling (a component of Hydrogen breath tests are commonly used to assess the dysfunction for most people with SCI). Clinically, complica- pathophysiology of clinical GI disorders. As there is no tions of LUT dysfunction remain the leading cause of 271 bodily source of hydrogen other than that produced by rehospitalization among people with SCI. bacterial metabolism in the cecum, an increase in hydrogen expiration following carbohydrate administration indicates Assessment of lower urinary tract function following the arrival of the nutrient bolus in the cecum, and can be experimental SCI used as a measure of OCTT.195–197 Using this test, individuals SCI induces profound changes in bladder innervation, parti- with SCI show significantly delayed OCTT compared to cularly afferent, circuitry,206,217,272,273 morphology209,213,274 controlsFan effect that is more pronounced in quadriplegic and structure,275–278 all of which likely contribute to neuro- than paraplegic patients.198 Although this technique has also genic bladder. We limit this discussion to functional assess- been validated in feline, canine and rodent models,195,199,200 ments only, with an emphasis on tests used in clinical SCI. it has not yet been used in experimental SCI. However, it Although an animal model of cauda equina/conus medullaris may prove to be a useful outcome measure to assess GI injury (lumbosacral ventral root avulsion in rat) has been function following SCIFat both the bench and the bedside. recently developed,279,280 available experimental data describe

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Figure 7 Innervation of the lower urinary tract (LUT). The LUT is comprised of the bladder, urethral sphincter and urethra. The LUT receives the bulk of its innervation from three nerves. The hypogastric nerve carries sympathetic innervation to the LUT; contributing spinal nerves exit the spinal cord (SC) between L1 and L2. Muscle activity for storage is mediated by a-AR expressed in the trigone, bladder neck and urethra (excitatory), and by b-AR expressed in the bladder dome (inhibitory). The pelvic nerve contains parasympathetic input originating in the sacral cord (L6–S1 in rat407) and controls micturition via cholinergic muscarinic receptors (mAChR) expressed throughout the LUT. The human pudendal nerve exits the sacral SC, and provides somatic innervation to the striated muscles of the external urethral sphincter; in rats, the pudendal nerve originates in the L6–S1 cord. In addition to their efferent function, each of these nerves carries afferent input from the LUT. Information about bladder distension is carried by mechanosensitive afferents (Ad, C) found primarily in the pelvic nerve. These afferents signal the coordinated switch between storage and micturition. The pudendal and hypogastric nerves mostly contain nociceptive afferents, which are not depicted here. Abbreviations: AR, adrenergic receptors; DR g, dorsal root ganglion; EUS, external urinary sphincter; g, ganglion; IM g, inferior mesenteric ganglion; L, lumbar spinal cord; mAChR, muscarinic cholinergic receptors; n, nerve; nAChR, nicotinic cholinergic receptors; NO, nitric oxide; P2X, purinergic receptor; S, sacral spinal cord; ( þ ) denotes excitatory synapses; (À) denotes inhibitory synapses.

bladder dysfunction following supraconal (not infraconal) SCI. thral, conscious or unconscious, cystometry in experimental Although SCI-induced LUT dysfunction has been well-char- animals is essentially similar to clinical cystometry: the acterized in experimental animals, few studies have included bladder is filled with saline while recording intravesical LUT assessment as an outcome measure in testing therapies for pressure to examine the relationship between bladder SCI (Table 9). volume and pressure during filling and micturition. Detrusor hyperreflexia and detrusor–sphincter dyssynergia create a similar urodynamic pattern in rats209,237,218 and Cystometric urodynamic analysis humans206,269,282,283 with suprasacral SCI. Post-injury cysto- The most common method for assessing LUT function in metrograms are characterized by increases in volume thresh- experimental animals is cystometric urodynamic analysis. old for inducing micturition, pressure during micturition, Cystometry can be performed using a urethral or transvesical volume expelled during micturition and residual volume catheter implanted into the bladder dome: although the after micturition. In addition, spinal-cord-injured rats and latter method is more invasive, it does not partially obstruct humans often exhibit nonvoiding detrusor contractions the urethra and permits EMG of the external urethral during bladder filling, a hallmark of detrusor hyperreflexia. sphincter (see ‘External urinary sphincter electromyogra- Rats assessed via conscious transvesical cystometry 2–3 phy’) to be performed concurrently. Cystometry is routinely weeks after complete thoracic (T8–T10) spinal transection conducted in both conscious and anesthetized animals, with had larger volume thresholds for micturition (1.43 versus the caveat that anesthesia does affect micturition reflexes,281 0.34 ml), larger micturition pressures (48 versus 26 mm Hg), particularly in animals with chronic SCI.235,236,257 This increased voided volumes (0.72 versus 0.31 ml) and in- undesirable effect may be partially addressed by reducing creased residual volumes (0.71 versus 0.03 ml) compared to the dose of anesthetic.236 Whether transvesical or transure- uninjured controls.218

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Table 6 Overview of studies characterizing or targeting lower urinary tract dysfunction after experimental SCI

Technique used Species Injury model Time range post-injury References

Cystometry Rat T8–T10 contusion p1 week 209–211 2–4 weeks 209,211,212 8 weeks 210,213 T8–T11 Tx Immediate 214 24 h 215,216 p1 week 217 2–4 weeks 209,214,216–234 4–8 weeks 225,231,235–245 8–12 weeks 232,246,247 T12 heat injury 1 month 248 L3/4; L6/S1 Tx Immediate; 2–5 weeks 214 Rabbit T10 Tx 1, 2, 7–21 days 249 Cat C6–T1 Tx 10 weeks 250 T1 clip compression 2–3, 5–7 weeks 251 T8 Tx; T8 contusion 3 weeks 252 T10 d. fun. Tx Immediate 253 T10–T12 Tx p24 h 254 2–4 weeks 255,256 4–8 weeks 255–261 6–12 months 257,260 Dog T8–T11 Tx 1–8 weeks 262 1–8 months 263,264

External urinary sphincter EMG Rat T8 contusion p1 week 209,213 2 weeks 209 6–8 weeks 210,213 T8–T9 Tx Immediate 214 2–4 weeks 209,214 4–6 weeks 214,241,243 T8–T11 Tx 2–4 weeks 219 4–8 weeks 236,237,239,240 8–12 weeks 246 L3/4; L6/S1 Tx Immediate; 2–5 weeks 214 Rabbit T10 Tx 1, 2, 7–21 days 249 Cat C6–T1 Tx 10 weeks 250 T1 clip compression 2–3, 5–7 weeks 251 T9–T12 Tx 2–4 weeks 256 6–8 weeks 253,258,261 Dog T8–T10 Tx 1–8 weeks 251 1–8 months 264

Bladder volume Rat T8; T9/10 contusion p1 week 213,265,266 2–4 weeks 265–267 4–6 weeks 265,266 T10 Tx 0–20 days 217 Cat T8 Tx; T8 contusion 2 weeks 252

Videofluoroscopy Cat C6–T1 Tx 10 weeks 250 T1 clip compression 2–7 weeks 251 Dog T8–T9 Tx 1–8 weeks 262

Pressure recording (c. spongiosus) Rat T9/10 contusion p1 week; 2–4 weeks 266

Abbreviations: compr., compression; c. spongiosus, corpus spongiosus; d. fun., dorsolateral funiculus; EMG, electromyography; Tx, transection. Studies have been classified by the technique used to assess lower urinary tract (LUT) function. Studies employing a combination of techniques are referenced under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.

Preclinical urodynamic analysis has been used extensively a few experimental studies testing regenerative, plasticity- to characterize mechanisms of and identify therapeutic promoting or protective treatments for SCI have incorpo- candidates for bladder dysfunction following SCI (Table 6). rated urodynamic analysis as an outcome measure (Table 9). Such experiments continue to be informative even after In one such study, rats that received transplants of treatments enter clinical practice: for example, rats were immortalized neural stem cells at the site of thoracic SCI recently treated with botulinum-A to examine the effects of (T8 contusion) exhibited reduced micturition pressure and acute versus delayed therapy following SCI.238 To date, only reduced residual urine compared to untreated controls.284

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Two subsequent studies in the same injury model demon- intrathecal chondroitinase treatment reduced the volume strated that transplantation of neural precursor cells285 or of urine that could be manually expressed in rats with genetically modified fibroblasts286 accelerated recovery from moderate thoracic SCI. bladder areflexia and reduced micturition pressure and the An attractive alternative to estimating bladder function by number of nonvoiding detrusor contractions. expressed urine is transabdominal ultrasound, which has been recently used in rat SCI.265 In this study, a handheld External urinary sphincter electromyography digital ultrasound imaging system was used to measure In both clinical and experimental assessment of SCI, external bladder volume following severe thoracic (T10) contusion. ± urethral sphincter EMG (EUS EMG) can be performed in Bladder volume was 3.51 0.47 ml (compared to ± conjunction with cystometry to provide a direct measure- 0.089 0.04 ml in uninjured rats) on day 4 post-injury and ± ment of detrusor–sphincter dyssynergia. Wire electrodes are decreased to 1.83 0.50 ml by day 8; it did not change inserted into the muscle of the EUS, and EUS activity during significantly for the duration of the experiment (46 days). bladder filling and micturition is recorded. The data are The authors found that ultrasonic measurements of bladder typically expressed as mean EMG activity, mean EMG power volume were comparable to estimates based on manual frequency, mean EMG-spiking activity (ESA) and durations expression of urine. of activity or contractions.213,236 Also, the relationship between EUS EMG amplitude and detrusor contractions is Advantages/disadvantages. Measurements of volume of ur- examined to determine the extent of coordination between ine per micturition represent a cost-saving alternative for detrusor and sphincter activities. Changes in EUS EMG monitoring bladder function, but are tedious and lack throughout the micturition cycle can be examined by power precision. Transabdominal ultrasound is noninvasive, less spectrum analysis using the fast Fourier transform algo- stressful than other methods, does not disrupt concomitant rithm.213 motor and sensory testing and permits assessment in the In rats209,213,237 and humans206,269,287,288 with suprasacral same animals throughout the recovery period. As this is a SCI, EUS EMG reveals sphincter activity that is not associated common clinically used test, it could be recommended for with detrusor contraction or micturition. In rats, the more frequent use in animal experiments. This method will micturition-induced increase in external sphincter activity require some investment in technology and training. (dESA) is lost following thoracic SCI: as dESA is negatively correlated with SCI severity, it can be used as an index of Videofluoroscopy 213 detrusor–sphincter dyssynergia. Other intriguing indices In videofluoroscopy, the bladder is filled with radio-opaque of bladder-sphincter synergy have been identified by analyz- medium and imaged by X-ray video recording. This ing fractal dimensions and power spectra of EUS EMG and approach is commonly used to evaluate a variety of organ 239,240 cystometrograms. Akin to urodynamic analysis, EUS functions in the clinical setting. Fluoroscopy has been used EMG has been used as an outcome measure in mechanistic in preclinical investigations of electrical stimulation to studies of LUT dysfunction in experimental SCI (Table 6). improve bladder function following SCI (Table 6). These However, this technique is rarely included in preclinical studies characterize the effects of sacral spinal stimula- studies that assess the functional benefits of therapies for SCI tion,262 direct bladder stimulation,251,290 and more recently, (Table 9). neuroprosthetic microstimulators targeting the bladder wall and plexus291,292 in dogs and cats with SCI. Advantages/disadvantages (Cystometric urodynamic analysis and External urinary sphincter electromyography). Cystometry and Advantages/disadvantages. Videofluoroscopy is a clinically EUS EMG applied in combination definitely represent the relevant technique and provides useful information for most informative and clinically relevant assessment of LUT specific applications (those mentioned above) in larger function following SCI. However, these are invasive, techni- species; however, it has never been used in rodent SCI. It cally demanding procedures that can only be performed at a also requires specialized equipment and expertise that is not single time point (that is, at the end of the experiment). likely to be available to most investigators. Laboratories that lack expertise in these techniques may wish to adopt another method of monitoring bladder Corpus spongiosum pressure recording function after SCI. Another method of examining LUT function that has been recently developed involves telemetric monitoring of pres- Functional bladder volume sure within the corpus spongiosum of the penis (CSP).266,293 In rats, increases in bladder volume are proportional to Traditionally used to study sexual function in experimental severity of SCI.213,274 Bladder volume is estimated by animals294–296 (see ‘Sexual function’), CSP pressure has been measuring the volume of urine expressed at micturition, recently validated for assessing LUT function in spinal-cord- either by using a metabolic cage267,284–286 or by measuring injured rats.293 In this study, the authors found that volume the volume of urine that can be manually expressed.289 of urine expelled per micturition was highly correlated with Bladder volume has been used as an outcome measure in micturition duration recorded by CSP pressure: thus, tele- experiments testing chondroitinase (a bacterial enzyme) as a metric CSP pressure monitoring can provide information regenerative therapy for SCI.289 In these experiments, about voiding volume and frequency of micturition in freely

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 19 moving, conscious animals throughout their recovery from requires the coordination of both the somatic and sympa- SCI. At 21 days after thoracic (T10) contusion, rats with thetic nervous systems. telemetric pressure monitoring had micturition CSP pressure The autonomic nervous system also has a role in the waveforms that were increased in duration, mean pressure maintenance of reproductive capacity, although there is and peak frequency compared to those observed before comparatively little research in this area.319 The activity of SCI.266 the epidydimis, whose main functions include sperm transport320,321 and fluid resorption,319 is primarily regulated by the sympathetic nervous system, with particularly Advantages/disadvantages. Pressure recording from erectile important innervation arising from the inferior mesenteric tissue is technically challenging, but has the benefits of ganglion.320,321 being able to be used chronically and in unanesthetized, freely moving animals. It is of particular interest for outcome assessment post-SCI because it allows for simultaneous Clinical impact of sexual dysfunctions following SCI recording of micturition and erection eventsFboth of which Low sexual satisfaction and sexual dysfunction are both well are perturbed following SCI. documented after SCI, and the resolution of these problems has been identified as a high priority.9,322 The disruption to autonomic circuits following SCI can result in a number of Sexual function different sexual changes and the sexual function subgroup of the ASIA/ISCOS working group is currently developing Innervation of the pelvic organs and genitalia international autonomic standards for documenting these 323 The autonomic innervation of the pelvic organs is essentially changes. Here we focus primarily on changes in sexual similar across mammalian species, and generally similar function rather than reproductive function. The effect of SCI between males and females.297 Here we review only the most on sexual function is highly dependent on injury level and pertinent features of tissue innervation in the pelvic organs the most commonly affected sexual responses are arousal and genitalia (Figure 8). More detailed reviews can be found and orgasm. elsewhere.298–300 Individuals with upper motor neuron lesions, and pre- served S2–S5 roots, generally have preserved reflex genital arousal, as the reflexes mediating erection are located in the The role of the autonomic nervous system in sexual function spinal cord. However, these individuals generally are unable Normal sexual function requires a combination of local to initiate genital arousal psychogenically. On the other spinal reflexes and descending cortical control.300,301 Genital hand, lower level SCI (infraconal or cauda equina) tends to arousal is a combined neurovascular and neuromuscular disrupt reflex vasocongestion, but can leave sympathetically response that can be initiated reflexively or psychogenically. mediated psychogenic arousal intact.306,307 These psycho- In both sexes, reflex sexual arousal results from increased genic erections are comparable in duration, rigidity and parasympathetic activity, which causes nitric-oxide- tumescence to the preserved reflex erections in men with mediated ,302–304 accompanied by inhibition of higher lesions.306 sympathetic adrenergic activity. Together these two changes Many women with SCI maintain the ability to reach lead to an increase in genital blood flow, engorgement of orgasm. However, men often have difficulty maintaining erectile tissues and intracavernous pressure increase and, erection, ejaculating and sensing orgasm. As a result, male in women, lubrication of the vaginal surface. Unlike reproductive function is significantly affected by SCI. Penile reflex arousal, psychogenic arousal appears to be facilitated vibratory stimulation and electroejaculation have been used by the sympathetic nervous system.305–308 In the male to successfully manage infertility in many cases.324–326 Most rat, and probably also in humans, the contraction of the women maintain reproductive capacity, and are able to somatic striated perineal muscles (bulbospogiosus and become pregnant and undergo normal pregnancy after a ichiocavernosus) also contributes to penile rigidity during short period of amenorrhea acutely after SCI.327,328 erection.309–312 AD must also be included in the discussion of sexual Sexual climax appears to be mediated by a spinal reflex:313 function, as it can be triggered by sexual activity and sperm in response to genital stimulation, anesthetized, acutely retrieval in those with injuries above T5.329–334 During spinalized rats show a urethrogenital response similar to that periods of AD, blood pressure can reach levels that are seen in human sexual climax.307,314–316 This reflex includes potentially life threatening. However, despite these extreme clonic contractions of the perineal muscles, rhythmic firing blood pressure levels, the symptoms of AD are not always of the cavernous nerve, penile erections and ejaculation in subjectively detected by the individual (termed silent AD).335 the male, and rhythmic vaginal and uterine contractions in Therefore, it is imperative that blood pressure be assessed in the female.316 This response is mediated by efferent output both private sexual activity and during sperm retrieval from hypogastric and pelvic nerves, suggesting that both procedures to assure that patients are not at risk.335 parasympathetic and sympathetic nervous systems are Interestingly, although AD interferes with sexual activity involved.317 With regards to the autonomic nervous system, for some individuals, the symptoms of AD during ejaculation itself is considered to be a sympathetically sexual activity can also be perceived as pleasurable or mediated event;318 however, normal anterograde ejaculation arousing.333,336

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 20

Figure 8 Innervation of the pelvic organs and genitalia. In the pelvic organs and genitalia there are three main tissue types: secretory, erectile and striated muscle. The majority of the autonomic innervation to these tissues comes from the bilateral pelvic ganglia (PG), which contains both sympathetic and parasympathetic neurons. Parasympathetic preganglionic neurons originate in the sacral cord (S2–S4 in humans; L6–S1 in rodents) and travel in the pelvic nerve to the PG. Sympathetic innervation originates in the lumbar cord (L1–L2) and travels via the hypogastic nerves to innervate the PGFin rodents, sympathetic nerves also travel to the PG via the pelvic nerve, which is mixed sympathetic and parasympathetic. Unlike human PG, which form a diffuse plexus on either side of the prostate (men) or cervix (women), rat PG are more condensed, and form two true ganglia. In both sexes, the largest nerve exiting from the PG is the cavernous nerve (also called penile nerve in males). In humans, somatic innervation of the striated perineal muscles, which include the ischiocavernosus, bulbocavernosus and levator ani, originates in the sacral cord (S2–4), whereas in the rats, this is shifted rostrally (L6–S1). Afferent information from the pelvic organs is relayed to the spinal cord via the ‘genitospinal’ nerves (pelvic, hypogastric and pudendal; for simplicity, only the pelvic nerve is illustrated here) and sensory pathways ascend bilaterally in the dorsal quadrant of the spinal cord.408 Abbreviations: AChR, cholinergic receptors; DR g, dorsal root ganglion; g, ganglion; IM g, inferior mesenteric ganglion; n, nerve; nAChR, nicotinic cholinergic receptors; NE, norepinephrine; NO, nitric oxide; NPY, neuropeptide Y; ( þ ) denotes excitatory synapses; (À) denotes inhibitory synapses.

Assessment of sexual response and reproductive function following recovery of autonomic circuits after injury.266 Spinal transec- experimental SCI tion models have been used to study the neurophysiology of Although the subjective responses from human studies are spinal sex reflexes in the absence of supraspinal control,316 indispensable to fully elucidate sexual functioning following and to study infertility post-SCI.339–341 However, sexual SCI,337 physiological data can be obtained from experimen- function has been rarely used as an outcome measure in tal animal models regarding normal sexual function338 and testing therapies for SCI (Table 7). Like human research,

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 21

Table 7 Overview of studies characterizing or targeting sexual dysfunction after experimental SCI

Technique used Species Sex Injury model Time range post-injury References

Behavioral scoring Rat M Mid-T Tx 28–52 days 342 T8–T9 Tx 1–7 days 343 T6 Tx 3, 4 weeks 344 Pressure recording c. cavernosus M T8–T9 Tx 2 weeks 345 c. spongiosus M T10 contusion p1 week, 2–4 weeks 266 Doppler flowmetry F T10 Tx 6–8 weeks 346 Epididymal sperm transport M T9 contusion 10 days 347

Abbreviations: c. cavernosus, corpus cavernosus; c. spongiosus, corpus spongiosus; Tx, transection. Studies have been classified by the technique used to assess sexual function. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.

experimental animal research has been dominated by the conjunction with other physiological measures, such as study of male sexual function, and there are comparably few EMG recording. Together, these two techniques have been validated experimental animal models of female sexual recently used as outcome measures for assessing the effects of function.348 This section focuses on functional assessments pharmacological manipulation to facilitate penile reflexes of autonomically mediated components of sexual and and ejaculation after experimental SCI.344 Similarly, the reproductive function, with a focus on those that have been combination of visual scoring and blood pressure recording used in experimental SCI. following penile reflex stimulation in a rodent model of SCI could be used to address the important clinical issue of vibrostimulation-triggered AD in men undergoing fertility Ex copula visual scoring treatment (JAI, LMR and AVK, unpublished observations). Visual scoring has long been used to assess sexual function in experimental animals. This semi-quantitative technique includes observation, grading and quantification of sexual Corpus spongiosum and cavernosus pressure recording arousal and copulatory events (mounts, intromissions and During erection, dilation of arteries and erectile tissue ejaculations in the male; lordosis in the female animals).349 relaxation increase blood flow and result in increased Ex copula tests have been primarily used in experimental SCI intrapenile pressure. Pressure recording of the corpus models, as functional copulatory behavior is unrealistic cavernosum (CC) or corpus spongiosum (CSP) has been used given the associated motor deficits. Animals are generally to study sexual function and erectile function in intact tested in a supine position with their legs and torso rats,294,355,356 and is the most common assessment of erectile restrained by straps.350 function used in preclinical trials.356 In this technique, the To test reflex sexual function, penile reflexes are elicited by CC or CSP is catheterized using a polyethylene tubing, or a the retraction of the penile sheath and light pressure on the hypodermic needle attached to tubing, connected to a base of the penis,351 mechanical stimulation of the ure- pressure transducer.345 The parameters that are commonly thra316,352 or by electrical stimulation of the dorsal penile measured include the baseline, peak and plateau pressures, nerve.343 The stereotyped responses occur in clusters and total area under the curve, as well as erection latency and include erections (reddening of the glans), cups (flaring of total number of erections.356 These outcome measures have the glans into a trumpet shape) and flips (anteroflexions of been used to characterize the sexual response changes that the glans of varying lengths).351,353,344 After SCI, these reflex occur after SCI and the effectiveness of drugs in restoring erections occur more quickly and more frequently than in normal responses. uninjured animals.342 Recent validation in rats with SCI showed that CSP Visual scoring can also be used to investigate psychogenic pressure recording is a reliable method to evaluate erectile erectile function. Penile reflexes triggered by central stimula- function over extended periods of time in conscious and tion of the medial preoptic area (a brain region with a well- freely moving animals as well as in restrained animals.266,293 established role in the facilitation of sexual behavior) Akin to previous reports using reflex erection tests on SCI revealed that the capacity for centrally mediated erections rats,311,351 SCI rats exhibited shorter time to first observed is preserved in a rat model of cauda equina injury.350 As erectile event compared to baseline values.266 CSP pressure erectile function has been traditionally associated with recording was sensitive to early changes in SCI rats: the parasympathetic activation, this research revealed that the number of CSP pressure peaks increased in SCI rats as early as presence of thoracolumbar sympathetic activity is sufficient 1 day post-injury, even though at this time the total number to mediate erection, and likely forms a component of normal of erectile events was not different from baseline values.266 erectile function.350 Similar results have also been found Furthermore, SCI rats had many more CSP pressure peaks per clinically.354 erectile event, revealing that although erections may be Like many of the other assessment methods described qualitatively similar after SCI, their physiology may be herein, visual scoring is most valuable when used in significantly altered.266 Pressure recording has been recently

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 22

used as an outcome measure for evaluating the effectiveness in invasive and terminal preparations. It is conducted in of pharmacological manipulation to restore erectile capacity anesthetized animals, as the recording is very sensitive to after SCI.345 movement.

Advantages/disadvantages. Described above; see Urinary Epididymal sperm transport assay bladder function (‘Assessment of lower urinary tract function Unlike the techniques described above, which assess sexual following experimental SCI’ and ‘Corpus spongiosum pres- function, this assay is utilized to assess reproductive function sure recording’). following SCI. Epididymal sperm transport is assessed by labeling of spermatozoa followed by quantification of 347 Perineal muscle electromyography labeled sperm throughout the length of the epididymis. Physiological responses and the role of perineal muscles can This technique offers an indirect way to assess the preserva- be measured during sexual behavior using EMG recording. tion of sympathetic innervation to the epididymis, as sperm This technique was used in the discovery of the urethrogen- transport through the epididymis is sympathetically 320,321 ital reflex, a model used to study human sexual climax.316 In mediated. Following SCI, rats show decreased epididy- 347 the men, wire electrodes are placed in the bulbospongiosus mal sperm transport. muscle, which is attached to the CSP; in women, this recording is usually taken from the smooth muscle of the Advantages/disadvantages. This assay is a terminal prepara- vagina.316 In both sexes, mechanical stimulation of the tion that demands some level of technical skillFfor both the urethra elicits clonic contractions of the perineal muscles injection of the label and the epididymal dissection. and rhythmic cavernous muscle activity, with expulsion of the urethral contents in the men.316 This technique has been validated in uninjured rats as well as spinalized, anesthetized Thermoregulatory function animals.316,357 Autonomic control of thermoregulatory effectors The thermoregulatory role of the autonomic nervous system Advantages/disadvantages. Like EMG in other systems, is primarily mediated by the sympathetic nervous system perineal EMG is generally a terminal preparation and is and its vasomotor, sudomotor and pilomotor effectors. conducted on anesthetized animals. However, it offers a Although each of these effectors can also be activated by readily quantifiable measure of the sexual response. emotional stimuli, for the purposes of this review, we focus only on their thermoregulatory function. The preganglionic Flowmetry neurons of these effectors are cholinergic, and lie in the As signs of female genital arousal are quite subtle, experi- thoracolumbar cord (T1–L2); the ganglionic neurons are also mental research in this area relies heavily on physiological cholinergic, and lie in the paravertebral ganglia. recording. Vaginal photoplethysmography offers a reliable Vasomotor efferents that innervate cutaneous vascular clinical measurement of vaginal blood volume and pulse beds regulate the amount of heat that is dissipated to the 307,358–360 amplitude. Experimentally, vaginal blood flow surrounding environment. Unlike the visceral vasoconstric- recording by laser Doppler flowmetry has been recently tors, cutaneous vasoconstrictors are not under strong established as a method to investigate sexual arousal in baroreflex control, but are strongly affected by core tem- 348,361,362 rats. In this technique, capillary blood flow is perature changes. Some areas of the skin are more specialized measured by a surface probe, placed on the ventral surface to perform thermoregulatory roles; areas with arteriovenous of the vagina or clitoral glans, which is connected to a anastamoses allow for quick transfer of blood from the 346,348,361 flowmeter. Using a bipolar hook electrode, electro- arteries to veins. Although the location of anastamoses varies stimulation is applied to the clitoral nerve or pelvic plexus to from species to species, their function is similar. 361 elicit a vascular response. In contrast to most sympathetic efferents, sudomotor After suprasacral SCI, unlike male rats, female rats efferents are cholinergic. When activated, these neurons act exhibited decreased response to electrostimulation; only via muscarinic receptors to increase sweat gland secretion. In 50% of animals showed increased clitoral blood flow, and humans, there are two types of sudomotor neurons, apocrine 346 even then, the increase was weak and nonsustained. In or eccrine. Eccrine glands (present over the entire skin contrast, uninjured female rats showed immediate and surface, with especially high levels on the palms, soles, face sustained increases in both clitoral and vaginal capillary and axillae) are primarily used for diffuse thermoregulatory 361 blood flow. Recently, this technique has been used as a sweating; apocrine glands are associated with hair follicles, functional outcome to evaluate the effectiveness of pharma- and are present in the genital, axillary and mammary cological agents to restore sexual arousal following experi- regions. Unlike humans, rats only have apocrine glands, 346 mental SCI. present on the plantar and palmar paw surfaces. These glands are activated by psychogenic rather than thermal Advantages/disadvantages. This technique offers a new way stimuli, and do not have a role in thermoregulation.363 to investigate the underrepresented issue of female sexual Pilomotor neurons are noradrenergic, and innervate dysfunction following SCI. Flowmetry itself is noninvasive, piloerector muscles, which are found throughout the hairy but it is often used in combination with nerve stimulation, skin of experimental animals and skin of humans. When

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 23 these muscles contract, they raise their associated hairs and tion, profuse sweating can contribute to a lowering of body ultimately augment insulation. The rostrocaudal innerva- temperature, which can result in hypothermia.374 tion pattern of piloerector muscles is similar to that of the sensory dermatomes. Assessment of thermoregulatory function following experimental SCI In this section we focus primarily on functional tests of Clinical implications of thermoregulatory dysfunction after SCI thermoregulatory control that are currently employed in SCI can disrupt the descending sympathetic input, leaving experimental SCI research. These studies most commonly large areas of skin and associated blood vessels, sweat glands aim to characterize the thermoregulatory changes that occur and hair follicles disconnected from supraspinal sympathetic after SCI, and a small number of them test approaches to control.364 As a result, sympathetic mechanisms do not mitigate these dysfunctions (Table 8). Molecular approaches respond adequately to adjust core temperature and maintain to thermoregulation are beyond the scope of this review and 377,378 equilibrium. The resulting thermoregulatory instability in have been recently reviewed elsewhere. both the acute and chronic periods can be severely debilitating. Core temperature recording Clinically, there are three main phenomena associated Core temperature recording provides a rough indication of with temperature dysregulation reported following SCI: the thermoregulatory capacity. Clinically, this type of poikilothermia, acute hyperthermia and exercise-induced information is easy to obtain, as it is part of routine care of hyperthermia.5 Poikilothermia is also called ‘environmental individuals following SCI. In animals, core temperature is fever’, but can refer to both the hypo- and hyperthermia also quite easily obtained using a rectal thermometer. experienced by individuals with SCI as a result of the Acutely after cervical and high-thoracic SCI, rat core ambient environment that they are exposed to. ‘Quad fever’ temperatures decline significantly and are vulnerable to 40,379 is used to refer to hyperthermia present in the first few weeks changes in ambient environmental temperatures. or months after SCI that is unrelated to infection or other Although the use of heating mats and raised environmental identifiable causes. temperatures can be used to maintain body temperature in 379 In general, individuals with high injuries are predisposed the acute setting, at 6 weeks post-injury core temperatures to more severe temperature dysregulation than those with remained significantly lower in SCI rats compared to 40 lower injuries.365,366 However, even individuals with low- preoperative levels. level SCI can exhibit up to a 50% reduction in whole-body sweating capacity compared to able-bodied controlsFa Advantages/disadvantages. Core temperature recording is reduction that correlates with their reduced skin surface minimally stressful for SCI animals with limited colorectal area for sweating.367,368 Although there is some evidence of sensation. The tools are easily available and the technique increased productivity of sweat glands above the injury as a itself requires minimal training. However, investigators must mechanism of compensation for decreased sweating below be aware that rectal probe insertion could induce AD, which the injury,369 individuals with SCI remain at a significant risk can alter core temperature.40,374 for heat illness due to increased heat storage.370 Heat storage is particularly problematic in wheelchair athletes,371 but Cutaneous temperature recording recent evidence suggests that precooling or cooling during Cutaneous temperature recording provides a simple indirect exercise can reduce the development of a high core way to investigate cutaneous vasoconstrictor activity. De- temperature.372,373 spite the fact that skin blood flow is important in heat loss As well as being at increased risk for thermoregulatory during exercise in individuals with SCI,380 there has been dysfunction, individuals with high-thoracic and cervical little research investigating cutaneous blood flow after injuries are also at risk for AD. During periods of AD, when experimental animal SCI.40 The skin of the tail is the most reflex sympathetic adrenergic vasoconstriction below the important thermoregulatory organ in the rodent, and has level of injury elevates systemic blood pressure, reflex been targeted in many investigations of thermoregulatory sympathetic cholinergic activity can also cause excessive function.381,382 Infrared thermography can be used to record sweating. In combination with thermoregulatory dysfunc- the surface temperature of the rat tail and hindlimb.40,383,384

Table 8 Overview of studies characterizing or targeting thermoregulatory dysfunction after experimental SCI

Technique used Species Injury model Time range post-injury References

Core temperature recording Rats C6/7 Tx 6 h 375 Infrared thermography T4 Tx 1 day–6 weeks 40 Flowmetry T2; T9 Tx 1 h–7 days 39 Microneurography T12–T13 Tx 0–72 h 376

Abbreviation: Tx, transection. Studies have been classified by the technique used to assess thermoregulatory function. Studies employing a combination of techniques are referenced under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 24

Following SCI, the skin temperature of the mid-tail and the SCI clinically, as it requires supraspinal input.390 However, hindpaw increased. These changes correlated with the no animal studies of SCI have included assessment of decrease in core temperature, suggesting that the two are sudomotor function as an outcome measure of autonomic potentially related.40 nervous system function.

Advantages/disadvantages. Cutaneous temperature record- Advantages/disadvantages. SSR is a noninvasive technique, ing is noninvasive, causes no stress to the animals and can but it has not yet been developed for use in animals. be used chronically. However, specialized equipment is required for both the data acquisition and analysis. Visualization of sudomotor function The visualization of sudomotor function is used in both Flowmetry clinical and in experimental research. Thermoregulatory Blood flow recording provides a noninvasive way to assess sweat testing with alizarin red has been used to define the sympathetic vasomotor pathways following SCI. Clinically, extent of peripheral neuropathies.391,392 Preliminary results this method has been used to show that individuals with show that this technique can also be used as a tool to map high SCI have reduced skin vasoconstriction in response to the preserved function of cholinergic sympathetic pathways cutaneous cold compared to controls, and reduced vasodila- in individuals with SCI.393 The starch iodine technique is an 385,386 tion in response to local heating below the injury. This analogous assessment that is used in rats to visualize sweat method is also used in experimental animal research, where gland function.394,395 ultrasound flow probes are surgically implanted around the artery, tissue or organ of interest, and flow is recorded using a Advantages/disadvantages. This technique is minimally in- flowmeter.39,382 Hong and colleagues have recently looked at vasive and the results could be easily translated between the organ system microcirculation, including the skin in the clinic and the experimental laboratory. However, in rodents, forepaw and hindpaw, after acute SCI at high and low it can only inform about the presence or absence of thoracic levels.39 In the acute period, there was a significant sympathetic innervation of the palmar and plantar surfaces. decrease in forepaw blood flow, which may be related to Furthermore, it does not reveal any information about the changes in regional peripheral vascular resistance.39 thermoregulatory response.

Advantages/disadvantages. Flowmetry can be minimally in- vasive (when recording from the skin surface), but requires a Conclusion significant investment in equipment. Although the equip- Historically, SCI was synonymous with paralysis, and the ment is quite sensitive to movements, caused by breathing ultimate goal of therapy was recovery of movement. Today, for example, there are ways to reduce these artifacts.39 SCI is more correctly recognized as a potential disruption of all nervous system functions, including motor, sensory and Microneurography autonomic. Although motor impairments are the most In vivo microneurography allows for the direct intraneural obvious manifestation of disability, they may not be the 387 recording of sympathetic neuronal activity. Changes in most catastrophic: injury to the spinal autonomic pathways sudomotor and vasoconstrictor activity can be recorded in results in functional deficits of the major organ systems, response to thermoregulatory stimuli, or nerve activity can manifesting as bladder, gastrointestinal and sexual dysfunc- be evoked to investigate the function of the peripheral tions, and disordered cardiovascular function, thermoregu- effectors. Microneurography has been recently used to assess lation and respiration. These aspects of autonomic whether therapeutic electrical stimulation can facilitate skin dysfunction are fast emerging as priorities in clinical 376 sympathetic nerve activity following SCI in rats. management of SCI.5,323 To address these priorities, discovery scientists must Advantages/disadvantages. Microneurography is both inva- incorporate autonomic assessments as outcome measures sive and technically challenging. However, it provides a in their experimental SCI research. Here we have reviewed a sensitive and direct measurement of sympathetic nerve wide variety of techniques that are available to evaluate activity. autonomic function in experimental animals, including some that have not yet been used in experimental SCI, but Sympathetic skin response appear to be feasible. For some laboratories, incorporating Clinical sudomotor tests have proven successful in the autonomic assessment may involve adding new techniques identification and localization of autonomic nervous system to their existing battery of outcome measures. For others, lesions.388 The sympathetic skin response (SSR) is a widely collaboration may be required to attain the necessary used clinical technique that assesses the integrity of expertise. In some cases, it might be necessary to add sympathetic cholinergic pathways. In humans, it is typically additional groups of animals to maintain scientific rigor. recorded from the palmar and plantar surfaces, areas rich in Whatever the practical considerations of incorporating eccrine glands, and measures changes in skin conductance in autonomic assessment, the gainFan improved understand- response to electrical nerve stimulation.389 SSR has been ing of autonomic dysfunction after SCIFis indisputably suggested as one way to assess autonomic completeness of worth the effort. There is an urgent need to include

Spinal Cord Autonomic assessment in experimental SCI JA Inskip et al 25

Table 9 Overview of studies testing protective or regeneration/plasticity promoting therapy for SCI

Technique used Species Injury model Time range post-injury Treatment tested References

Bladder Cystometry Rat T8/9 compression 2 weeks; 1 month Neural stem cell transpl. 284 (9 days after SCI) T8–T11 contusion 8 weeks Neural precursor transpl. 285 (9 days after SCI) Modified fibroblast transpl. 286 (9 days after SCI) 2 months Schwann cell transpl. 396 (1 h after SCI) Bladder volume T8/9 compression 2 weeks; 1 month Neural stem cell transpl. 284 (9 days after SCI) T8–T12 contusion 0–24 h; 1–8 weeks Neural precursor transpl. 285 (9 d after SCI) 8 weeks Modified fibroblast transpl. 286 (9 d after SCI) 0–100 days Intrathecal chondroitinase ABC 289 (for 2 weeks from the time of SCI)

Cardiovascular Autonomic dysreflexia Rat T4 clip compression 2, 6 weeks Anti-CD11d antibody and 397 (colorectal distension) methylprednisolone (singly and in combination; i.v.; 2, 24 and 48 h post-injury) 2, 6 weeks Anti-CD11d antibody 398 (i.v.; 2, 24 and 48 h post-injury) 5 weeks Anti-CD11d antibody 399 (i.v.; 6 h post-injury) 6 weeks Polyethylene glycol and 400 magnesium sulfate (i.v.; 15 min and 6 h post-injury)

Respiratory Phrenic nerve conduction Rat C2 hemisection 3–6 months OEC transpl. (at injury site) 141 High C hemisection 2 months 140 Diaphragm EMG C2 hemisection 3–6 months 141 Plethysmography T8 contusion 24 h, 7, 28, 35 days FGF infusion (at injury site) 401

Abbreviations: EMG, electromyography; FGF, fibroblast growth factor; i.v., intravascular; OEC, olfactory ensheathing cells; transpl., transplantation; Tx, transection. Studies have been classified by the autonomic system that they assess, as well as by the technique used to assess that system. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.

autonomic evaluations in experiments testing therapeutic on Repair Discoveries) for providing a supportive environ- (that is, regenerative or protective) agents after SCI, as there ment for our research. are very few experiments currently doing so (see Table 9). At present, treatments are moving to clinical trial without any animal data that might predict their effects on References 402 autonomic function. If we continue to neglect autonomic 1 Courtine G, Song B, Roy RR, Zhong H, Herrmann JE, Ao Y et al. function after SCI in the laboratory, the consequences for Recovery of supraspinal control of stepping via indirect people with SCI could be disastrous. propriospinal relay connections after spinal cord injury. Nat Med 2008; 14: 69–74. 2 Dunham W. Scientists move toward helping paralysis patients. Reuters, 1-7-2008; http://www.reuters.com/article/scienceNews/ Acknowledgements idUSN0428923520080107. 3 UCLA finds possible paralysis cure. Press TV. 1-9-2008; http:// We gratefully acknowledge the Heart and Stroke Foundation www.presstv.ir/Detail.aspx?id ¼ 38099§ionid ¼ 3510210. of BC and the Yukon (AVK and MSR), the Christopher Reeve 4 Sipski ML, Arenas A. Female sexual function after spinal cord injury. Prog Brain Res 2006; 152: 441–447. Paralysis Foundation (AVK), the Rick Hansen Foundation 5 Krassioukov AK, Karlsson AK, Wecht JM, Wuermser LA, Mathias (AVK and MSR), the Michael Smith Foundation for Health CJ, Marino RJ. Assessment of autonomic dysfunction following Research (JAI, LMR and MSR), the Canadian Institutes of spinal cord injury: rationale for additions to International J Rehabil Res Dev Health Research (JAI and MSR) and the National Science and Standards for Neurological Assessment. 2007; 44: 103–112. Engineering Research Council (LMR) for their support. We 6 Weaver LC, Polosa C. Autonomic dysfunction after spinal cord thank our colleagues at ICORD (International Collaboration injury. Prog Brain Res 2006; 152: 1–453.

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